We construct a simple fluorescent biosensor for single-molecule counting of flap endonuclease 1 (FEN1) based on ligase detection reaction (LDR) amplification-activated CRISPR-Cas12a. This biosensor exhibits excellent selectivity and high sensitivity with a detection limit (LOD) of 1.31 × 10 U. Moreover, it can be employed to screen the FEN1 inhibitors and quantitatively measure the FEN1 activity in human cells and breast cancer tissues, holding great promise in clinical diagnosis and drug discovery.
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